Structural Characterization of the High Affinity Glycopeptides of Glycam-1, a physiological ligand of L-selectin. The binding of the selectins (calcium-dependent lectin-like receptors) to their endogenous glycoprotein ligands mediates the initial interactions between leukocytes and endothelia. L-selectin is involved in the trafficking of leukocytes to inflammatory sites and to organized lymphoid organs. A glycosylated cell adhesion molecule (GlyCMA-1), derived from high endothelial venules of lymph nodes, is a soluble, physiological ligand for L-selectin. GlyCAM-1 appears to be a signaling molecule that interacts with lymphocyte L-selectin to induce a high avidity state of the integrin, LFA-1. GlyCAM-1 is a mucin-like glycoprotein with a heterogeneous array of oligosaccharides with 6' and 6 sulfated sialyl Lewis X typetermini. The polypeptide backbone of 133 amino acid residues has 10 clusters of contiguous hydroxy-amino acid residues (dyads, triads or a tretrad), suggesting that a closely packed ensemble of olgiosaccharides forms the biologically relevant ligand. We propose to identify the arrays of O-glycans using tandem mass spectrometry with collision-induced dissociation and Edman sequencing. Our preliminary studies using HPLC-electrospray ionization mass spectrometry (LC-ESI/MS) have shown that trypsin cleaves GlyCAM-1 between the two glycosylated regions. We will fractionate tryptic digests of GlyCAM-1 using L-selectin affinity chromatography. The oligosaccharides from each regional glycopeptide will be released by b-and 6- O-SO3-GlcNAc. The high affinity regional glycopeptides will be digested with an array of specific endoproteases to produce glycopeptides containing different combinations of the Ser/Thr clusters. The digests will be analyzed using LC-ESI/MS with selective ion monitoring (m/z204) to located the glycosylated fragments. These smaller glycopeptides will be fractionated by affinity chromatography using an immunoglobulin/L-selectin chimera. The oligosaccharides willbe released by b-elimination from the L-selectin binding glycopeptides. Using matrix assisted laser desorption ionization (MALDI) with collision induced dissociation (CID), we will characterize the O-glycans with respect to molecular weights, composition (in terms of Neu5Ac, Hex,deoxyHex, NexNAc, Hex-SO3 and HexNAc-SO3), sequence, branching and residue location of sulfate moieties. Oligosaccharides will be assigned to specific peptide loci using MALDI-CID. Spectra will be obtained with a tandem instrument. These last analyses will give i) the peptide sequence from the mass of the gas-phase de-glycosylated peptide and associated fragments; ii) the masses of the intact oligosaccharide chains; and iii) the peptide location of the oligosaccharide chains from the masses of the glycosylated peptide fragments.